We were the first to demonstrate that stimulation of IL-3 dependent murine mast cell lines with calcium ionophores, or by crosslinking high affinity IgE receptors (FcepsilonRI) with antigen or anti- IgE, induces the production of IL-3, IL-4 and other cytokines. We have also found that crosslinking of FcepsilonRI on two freshly isolated in vivo cell populations in non-immune mice, resting peritoneal mast cells and putative mast cell precursors in bone marrow, also induces cytokine secretion. This newly recognized ability of mast cells to secrete cytokines suggests a much wider role in inflammation than previously anticipated. This grant intends to extend our focus, to assess cytokine production in normal mast cells and determine how cytokine production relates to the state of mast cell differentiation both ontogenetically and phenotypically. First, we will test for qualitative differences in cytokine production induced by FcepsilonRI crosslinking of mast cell populations at distinct stages of differentiation, including mast cell precursors, mast cells differentiating during short vs. long term culture of bone marrow or spleen with IL-3, mature peritoneal mast cells, and mast cell lines differentiating into connective tissue phenotype. Second, we will test for quantitative and qualitative differences in the capacity of agents to induce cytokine production and/or regulate FcepsilonRI-mediated cytokine production by mast cells. We will explore the nature of FcepsilonRI crosslinking required and will also determine whether complement anaphylatoxins, and tachykinins, such as substance P, activate cytokine production. Since mast cells may express class II MHC molecules, we will determine whether mast cells activate T cells and whether T cells directly activate cytokine secretion by mast cells. We will extend our preliminary studies indicating that IL-3 markedly enhances the capacity of FcepsilonRI crosslinking to induce cytokine production, and test the regulatory effects of combinations of IL-3 with IL-4 and TGF-beta. Finally, we will evaluate the capacity of pharmacologic agents (which inhibit mast cell histamine secretion) to regulate cytokine secretion. We will determine whether any of these agents differ from FCepsilonRI crosslinking in their capacity to induce a specific spectrum of cytokines, as FcepsilonRI crosslinking induces the production of IL-3, IL-4, IL-5, IL-6 but not IL-2 and interferon-gamma. These experiments should provide a fuller understanding of the potential major role of mast cell precursors in immunologically mediated inflammation.